Structural biology provides an important tool for the detailed characterization of proteins at the molecular level. This molecular approach can lead to a more complete understanding not only of a protein itself, for example, but also helps characterize the interactions between a ligand-binding protein and its known ligands and/or putative binding partners. The nuclear hormone receptor farnesoid X receptor (FXR) functions as a bile acid sensor by responding to physiological levels of a variety of bile acid ligands and coordinating the control and maintenance of lipid homeostasis. Elucidation of the three-dimensional structure, and in particular, the structure of the ligand binding domain involved in binding bile acids, can assist in studies of the function and physical properties of FXR.
An essential function of the liver and the intestine in vertebrates is to maintain lipid homeostasis within the body through tight regulation of the acquisition, synthesis and metabolism of cholesterol (Chawla et al. (2000). “Don't know much bile-ology”. Cell. 103, 1-4). Excess cholesterol is either converted into bile acids in the liver, or undergoes biliary excretion in the intestine and is disposed of in the stool (Chiang (2002) Bile Acid regulation of gene expression: roles of nuclear hormone receptors. Endocr Rev. 23(4), 443-63). The nuclear hormone receptor (NHR) farnesoid X receptor (FXR, also known as NRIH4) is involved in the regulation of both of these metabolic processes. FXR is expressed in the liver and intestine as well as other cholesterol rich tissues such as the adrenal gland. Knockout mice deficient in FXR expression display defects in bile acid (BA) homeostasis when exposed to dietary stresses, including elevated serum BA, reduced bile acid pools, and reduced fecal BA secretion (Sinai et al. (2000). Targeted disruption of the nuclear receptor FXR/BAR impairs bile acid and lipid homeostasis. Cell: 102(6), 731-44). In the liver, the rate-limiting step for the conversion of excess cholesterol into bile acids is catalyzed by the cytochrome p450 gene, cholesterol 7alpha-hydroxylase (CYP7A1). A second cytochrome p450 gene, sterol 12 alpha-hydroxylase (CYP8B) is a key enzyme for regulating the cholic acid (CA)/chenodeoxycholic acid (CDCA) ratio in bile acid biosynthesis (Kerr et al., (2002) Loss of nuclear receptor SHP impairs but does not eliminate negative feedback regulation of bile acid synthesis. Dev Cell. 2(6), 713-20; Wang et at (2002) Redundant pathways for negative feedback regulation of bile acid production. Dev Cell. 2(6), 721-31). In mammals these genes are indirectly regulated by FXR via the NHR homologue gene SHP (small heterodimer partner) (Lu et al. (2000). Molecular basis for feedback regulation of bile acid synthesis by nuclear receptors. Mol. Cell. 6(3), 507-15; Goodwin et al (2000). A regulatory cascade of the nuclear receptors FXR, SHP-1, and LRH-1 represses bile acid biosynthesis. Mol. Cell. 6(3), 517-26).
Physiological concentrations of specific BAs bind and activate FXR, the most potent being CDCA a major primary bile acid found in human bile (Makishima et al, (1999) Identification of a nuclear receptor for bile acids. Science. 284(5418), 1362-5; Parks et al. (1999). Bile acids: natural ligands for an orphan nuclear receptor. Science. 284(5418). 1365-8, and Wang et al. (1999) Endogenous bile acids are ligands for the nuclear receptor FXR/BAR. Mol. Cell. 3(5), 543-53). This activation enables FXR to act as a transcriptional sensor for bile acids (BAs), repressing the transcriptional expression of both CYP7A and CYP8B genes by increasing the levels of the inhibitory nuclear receptor SHE SHP is a promiscuous inhibitory heterodimer partner of NHRs that suppresses the transcriptional activity of a large number of NHRs. However, its ability to bind and inhibit the liver receptor homologue (LRH-1) a NHR required for CYP7A gene expression, indirectly allows FXR to exert its influence on cholesterol homeostasis (Lu et al., (2000), supra; Goodwin et al., (2000), supra). Additionally, BA activation of FXR positively regulates the expression of genes involved in the excretion and transportation of BAs including intestinal bile acid-binding protein (IBABP), bile salt export pump (BSEP) and canalicular multi-specific organic anion transporter (cMOAT) (Chiang (2002), supra). Thus, this receptor plays a key physiological role in the regulation of lipid homeostasis.
FXR belongs to a superfamily of ligand-inducible transcription factors involved in a wide array of biological functions including development, differentiation and homeostasis. The family members share two structurally-conserved domains; a central, highly conserved DNA binding domain (DBD) that targets the receptor to specific DNA sequences, termed hormone response elements, and a ligand binding domain (LBD) that binds small lipophilic hormones (Evans R M. (1988) The steroid and thyroid hormone receptor superfamily. Science. 240(4854), 889-95). The LBD functions as the regulating molecular switch. Binding of the appropriate hormone to the LBD causes a conformational change that results in the release of bound co-repressor proteins and the recruitment of co-activator proteins that culminates in the activation of transcriptional target genes. This regulation of NHR transcription factors by small lipophilic hormones makes this gene family an ideal target for chemical biology to identify novel chemical activators (Blumberg and Evans (1998). Orphan nuclear receptors—new ligands and new possibilities. Genes Dev. 12(20), 3149-55). FXR senses BA levels and mediates the repression of genes that convert excess cholesterol into bile BAs as well as the induction of BA transport genes makes FXR an attractive pharmaceutical target. The availability of potent synthetic agonists for FXR, and an understanding of how various binding agents interact with the ligand binding domain of FXR is a critical step required for the validation of FXR as a drug target and the elaboration of the functions of FXR.